The normal human antithrombin (ATIII) gene has been cloned and fully characterized in this laboratory. We have used the information gained from these studies to investigate the molecular spectrum of ATIII deficiency. We have also described a new amino acid mutation which, in the family studied, results in a dysfunctional ATIII with defective serine protease binding. This has allowed us to define the minimal size of the thrombin binding domain. We will introduce oligonucleotide-directed point mutations into this domain and express the cDNAs in E.coli. Each of the resultant mutant proteins will be studied for its ability to bind thrombin. These studies will thus define the most critical amino acids in the thrombin binding region and will allow us to predict naturally occurring ATIII variants that are likely to be encountered in clinical settings. In a more basic study, we will continue our investigations of mRNA processing using the 3' end of the ATIII gene as a model. We have already described two regions downstream of the poly A signal which function independently to direct cleavage of the primary mRNA transcript. We will undertake studies which we will introduce linker scanning and point mutations into these regions to define more precisely their role in mRNA processing. Studies are also proposed to determine the possible role of mRNA 3' ends in conferring tissue specificity. Our interest in eukaryotic gene regulation will be expanded to include studies of the role of c-myc and c-myb proto-oncogenes in Friend murine erythroleukemia cell (F-MEL) differentiation. Our previous work has shown that deregulated c-myc expression inhibits F-MEL differentiation. We will utilize various eukaryotic vectors to constitutively or inducibly express c-myc or c-myb antisense RNAs. The effect of stable expression of these constructs on cellular differentiation will be assessed. Cell lines which fail to differentiate because of constitutive c-myc expression will be useful in identifying transcripts which are regulated by the c-myc gene product.